Processes for chemical compounds

ABSTRACT

THE PRESENT PROCESS PROVIDES A METHOD FOR CONVERTING GERMINE TO THE 3,16 DIESTER THEREOF BY THE INITIAL REACTION OF GERMINE AND ACETONE UNDER ANHYDROUS CONDITIONS IN THE PRESENCE OF A HYDRATE-FORMING SULFONIC ACID PRODUCE AS A FIRST INTERMEDIATE GERMINE ACETRONIDE ORGANIC SULFONIC ACID SALT HYDRATE SUBSEQUENTLY NEUTRALIZING THE ACETONIDE SALT HYDRATE TO FORM GERMINE ACETONIDE, ISOLATING AND ESTERIFYING THE GERMINE ACETONIDE TO FORM THE GERMINE ACETONIDE DIESTER AND HYDROLYZING SAID ACETONIDE DIESTER UNDER ACIDIC CONDITIONS TO PRODUCE THE DESIRED GERMINE DIESTER.

United States Patent 3,632,811 PROCESSES FOR CHEMICAL COMPOUNDS Arthur E. Erickson, Cranford, N.J., assignor to Merck & (10., Inc., Rahway, NJ. No Drawing. Filed Nov. 27, 1968, Ser. No. 779,607 lint. Cl. C07d 33/48 U.S. Cl. 260-287 3 Claims ABSTRACT OF THE DISCLOSURE The present process provides a method for converting germine to the 3,16 diester thereof by the initial reaction of germine and acetone under anhydrous conditions in the presence of a hydrate-forming sulfonic acid to produce as a first intermediate germine acetonide organic sulfonic acid salt hydrate, subsequently neutralizing the acetonide salt hydrate to form germine acetonide, isolating and esterifying the germine acetonide to form the germine acetonide diester and hydrolyzing said acetonide diester under acidic conditions to produce the desired germine diester.

BRIEF SUMMARY OF THE INVENTION The present invention relates to an improved process for the preparation of germine diesters. More specifically, it relates to a selective method of preparing either 3,16 diesters or mono esters of germine in pure form and in high yield. Still more specifically, the invention relates to one step in the process of converting germine to its abovenamed diesters or mono esters which includes the reaction of germine with acetone to produce the important intermediate germine 14,15 acetonide in substantially quantitative yield.

DETAILED DESCRIPTION OF INV ENTION.

BACKGROUND The disease known as myasthenia gravis is one which affects a substantial portion of the population and is char acterized by fatigue and exhaustion of the muscular system marked by progressive paralysis of muscles without sensory disturbances or atrophy. It may affect any muscle of the body but especially those of the face, lips, tongue, throat and neck. One of the methods used in the past for the treatment of or the alleviation of the symptoms of this disease is the administration of drugs in an attempt to reverse at least temporarily some of the Wasting effects of the disease. Included among the drugs administered was a mixture of veratrum alkaloids. This particular mixture, although it appeared to have some salvatory effect, could only be used to a limited extent because of the variety of side effects attendant on the administration of the medication.

Germine is one of the alkaloids Which occur in the mixture of veratrum alkaloids referred to hereinabove. Recent studies carried out by Flacke and co-workers at the Harvard Medical School have shown that certain esters of germine, notably the 3,16 diacetate and the 3 monoacetate of germine were effective in alleviating skeletal muscle weakness in patients with myasthenia gravis without having the attendant side effects occasioned by the administration of the veratrum. alkaloid mixture.

One of the problems involved in the use of germine esters is the high cost of producing the pure material for administration. The starting material, which is the alkaloid germine, is only available by extraction from veratrum alkaloid mixtures or from other fermentation sources. For this reason, it is imperative, in converting germine to its mono or diesters, that the processing cost be as low as possible and that the yield of the ester be maximized. One of the prior art procedures involved the 3,632,811 Patented Jan. 4, 1972 conversion of germine to its 14,15 acetonide, acetylation of the acetonide and hydrolysis to the desired diester, the overall yield of which was severely limited by the low yield of germine acetonide formation. According to one such prior art method, germine is converted by treatment with excess hydrochloric acid to its hydrochloride salt which is then dissolved in a minimum volume of ethanol and then reacted with acetone to form the desired acetonide. Unfortunately, this method results in relatively low yields of the desired germine acetonide as Well as decomposition of the reactants with resultant contamination of the product. In order to further convert the germine acetonide to the desired germine ester, additional purification and loss of yield of intermediate product must be sustained in order to obtain the germine esters in high purity.

DESCRIPTION 22 CH3 11 12 17 OH 141516 1 U i 2 10: s OH ,----0 011 OH no- 5 The selective acetylation of the 3 and the 16 positions is a virtual impossibility without blocking some of the more readily acylable positions.

In accordance with my invention, germine is reacted with acetone under anhydrous conditions in the presence of an acid catalyst to form the 14,15 acetonide of germine, thus effectively preventing substantial acylation at positions other than 3 and 16 of the germine molecule. The present process differs from the prior art process in two important aspects. The reaction is carried out (1) under scrupulously anhydrous conditions and (2) in the presence of a hydrate-forming catalyst. The reaction is preferably carried out in a solvent for the reactants and for the hydrate-forming acid. Polar solvents are preferable and these include lower alkanols of from 1-6 carbons such as methanol, ethanol, propanol and the like, lower aliphatic esters, especially esters of C -C aliphatic carboxylic acids with C -C lower alkanols such as methyl acetate, ethyl acetate, propyl acetate, methyl propionate, ethyl propionate, propyl propionate and amyl acetate, N,N-diloweralkyl alkanoic acid amides such as dimethyl formamide, dimethyl acetamide or mixtures of said solvents with aromatic hydrocarbons as for example benzene, toluene, and xylene. Lower alkanols such as ethanol, propanol and butanol are preferred.

The acid catalyst used in the reaction of germine with acetone is an important feature of the process of my invention. The acid used must be one which readily combines with Water to form a stable hydrate, but is used in its anhydrous or dehydrated form. When used as one of the necessary reactants in the process of my invention, the acid serves a two-fold purpose. Thus, the acid serves as a catalyst for acetonide formation and as a dehydrat ing agent which reacts with the water formed as a result of the acetonide formation. The particular acid selected and the amount used depends on the ability of the acid to form a stable hydrate and must be used in sufiicient quantity to react with the water generated by the acetonide formation.

A class of acids which are particularly useful in the process of my invention are organic sulfonic acids. Especially preferred reagents are aryl sulfonic acids such as benzene sulfonic acid, p-toluene sulfonic acid, 2,4-dinitro benzene sulfonic acid, 3,5-dinitro benzene sulfonic acid, and a and 18 naphthalene sulfonic acids.

In accordance with one embodiment of my invention, the salt formed by reaction of germine and the selected anhydrous hydrate-forming acid is prepared in solution and mixed with an excess of acetone under anhydrous conditions. When this reaction is carried out in the preferred solvent, i.e. a lower alkanol such as ethanol, the reaction occurs readily at room temperature and, after a period of from a few minutes to about 18 hours, the formed germine acetonide acid addition salt separates from solution as a pure crystalline product in substantially quantitative yield.

The necessary reactants may be mixed in any order with or without added solvent. The three essential elements are germine, acetone, and a sufficient quantity of an aryl sulfonic acid to readily react with any water formed during the course of the reaction. In a preferred method of operation, the anhydrous aryl sulfonic acid and germine are mixed in a 1:1 molar ratio to form an anhydrous salt of germine. The anhydrous aryl sulfonic acid is preferably prepared just prior to the reaction with germine by the azeotropic drying of a requisite quantity of the selected aryl sulfonic acid in a minimum amount of benzene. Thus, for example, p-toluene sulfonic acid monohydrate is suspended in a small amount of benzene and the benzene distilled at atmospheric pressure until the water is removed by azeotropic distillation, leaving as a residue anhydrous p-toluene sulfonic acid dissolved in benzene. This p-toluene sulfonic acid is then reacted under anhydrous conditions with an equimolar amount of germine to form the anhydrous salt or germine and p-toluene sulfonic acid suspended in benzene. The benzene is then preferably removed by evaporation under reduced pressure leaving the substantially pure acid addition salt of germine as a crystalline residue.

The germine acid addition salt is then dissolved in a solvent which is miscible with acetone and to the solution acetone is added in at least a 1:1 molar ratio but preferably in excess beyond the stoichiometric requirement. The acetone reactant is added to the solution in an amount ranging between 1 to 100 moles of acetone per mole of germine salt reactant; preferably at least a 50 molar excess is employed. The reaction is carried out at ambient temperatures, although it can be conducted at temperatures from C. up to and including the reflux temperature of the reaction system. Thus, the reaction is preferably conducted at temperatures between 0 C. and 80 C. and most preferably from about 20-40 C.

Any organic solvent which is inert under the reaction conditions of acetonide formation may be employed. Solvents which are utilized preferably include those solvents which are polar in nature and in which germine acid addition salts will readily dissolve. These include alcohols, esters, N,N-dialkyl alkanoic acid amides, and other low molecular Weight, highly polar solvents of the type mentioned hcreinabove. It is essential for the success of the reaction that strictly anhydrous material be employed and that water be carefully excluded from the reaction mixture.

When the preferred conditions are employed, the germine acetonide acid addition salt forms readily and in a few minutes. In order to insure completion of reaction and a maximum yield of product, the reactants are stirred together at a temperature of from 20-40 C. for a period of from /2 to about 24 hours. The reaction time may be further extended up to about 48 hours without impairing the yield, but it is believed that no substantial increase in yield is produced by an extension of the reaction time since essentially quantitative yields are produced in a few hours. The product prepared by this method is essentially insoluble in the reaction mixture and the substantially pure crystalline germine acetonide acid addition salt separates and is recovered by any convient method, such as for example by filtration, centrifugation, or removal of the excess acetone and solvent by distillation under reduced pressure leaving the product as a residue.

The germine acetonide acid addition salt is then converted to the acetonide free base by reaction with will-- cient aqueous alkali to neutralize the acid thus decomposing the acetonide salt and freeing the acetonide as the base. In neutralizing the salt with aqueous alkali in order to form the acetonide free base, any aqueous alkaline solution may be used. In the event that strong bases such as sodium hydroxide, potassium hydroxide and the like are employed, it is preferred that dilute solutions of such bases be used in order to avoid decomposition of the germine by contact with strong concentrated alkali.

A preferable method of neutralizing the acetonide salt involves the use of aqueous solutions of weakly-alkaline materials such as ammonia, alkali metal carbonates and bicarbonates, including sodium carbonate, potassium carbonate, sodium bicarbonate, and the like. The neutralization reaction is conducted by adding together an aqueous solution of germine acetonide acid addition salt and sufficient aqueous alkaline solution to adjust the pH of the reaction mixture to 10. The neutralization is preferably carried out in a two-phase reaction mixture, which contains in addition to the aqueous phase a water-immiscible solvent for the acetonide free base. It is believed that use of the two-phase system tends to drive the neutralization reaction to completion by extracting the acetonide free base from the aqueous phase as it is formed. [Following the formation of the acetonide free base, the alkaline solution is extracted with a solvent for the acetonide which is insoluble in water. Solvents which are useful in extracting the desired acetonide are halogenated hydrocarbons such as methylene chloride, ethylene chloride, chloroform, carbon tetrachloride, and the like, alkanoic hydrocarbons such as benzene, toluene and xylene, lower aliphatic esters of lower aliphatic carboxylic acids as for example ethyl acetate, ethyl propionate, ethyl butyrate, methyl acetate, amyl acetate, and the like. The extract of acetonide free base is dried and the germaine acetonide recovered in any convenient manner such as by removal of the extracting solvent by evaporation under reduced pressure and leaving the germine acetonide as a residue in substantially pure form.

In converting germine acetonide to germine 3,16 diacetate, the acetonide is treated with a mixture of equal parts of acetic anhydride and pyridine at a temperature of 25 C. for a period of about 24 hours. Acetic anhydride is used in excess of the stoichiometric amount required theoretically for diacetylation of the germine acetonide. In carrying out this acetylation of the acetonide, a large excess of acetic anhydride may be used without affecting acetylation of any hydroxyl substituents other than the 3 and 16 hydroxyls. The ratio of acetic anhydride to the germine employed is between about 2 moles of acetic anhydride per mole of germine acetonide to 20 moles of acetic anhydride per mole of germine acetonide. A ratio of about 5 moles of acetylating reagent to 1 mole of germine acetonide is believed to produce an optimum result. After the acetylation reaction is complete, the excess acetylating reagent is preferably removed by evaporation under reduced pressure followed by washing of the acetylated product several times with a solvent of an anhydride such as dry benzene. The substantially pure germine 14,15 acetonide 3,16 diacetate is used directly in the step of hydrolysis to the desired 3,16 diacetate without further purification.

The germine acetonide diacetate is hydrolyzed in dilute aqueous acid at ordinary temperatures for a short period of time to produce germine 3,16 diacetate in excellent yield. In a preferable method of carrying out the hydrolysis, germine acetonide 3,16 diacetate is dissolved in dilute hydrochloric acid and allowed to stand at a temperature of from 30 C. for a period of for a few minutes to several hours, preferably about 30 minutes. After the acidic hydrolysis is complete, the solution is neutralized. In a preferable method of carrying out the neutralization, the pH is adjusted to about 8.5 with ammonium hydroxide. The germine diacetate produced by the acidic hydrolysis is then extracted from the neutral solution with a solvent, as for example, methylene chloride. Removal of the methylene chloride solvent by evaporation yields as a residue the product germine diacetate as substantially pure, colorless crystals melting at 229- 230 C. Recrystallization gives a high yield of high purity product.

The germine 3-monoacetate is produced using a slight variation of the above procedure. Thus germine 14,15 acetonide is acetylated by treating with excess acetic anhydride-pyridine mixtures for a somewhat shorter period of time at C., i.e. approximately 2 to 5 hours but preferably 3 /2 hours at 2030 C., for the acetylation reaction. The product obtained as a result is a mixture comprising predominantly the 3 acetate of germine 14,15 acetonide containing a small amount of the 14,15 acetonide of germine 3,16 diacetate. The germine diacetate in the mixture is completely converted to the 3-monoacetate by heating the mixture at reflux temperature in methanol for a period of 8 hours. Thus the entire product is converted to germine 14,15 acetonide 3-monoacetate. The hydrolysis of the acetonide of germine 3-monoacetate is carried out in a manner similar to that described for the diacetate. The substantially pure product obtained melts at 219-220 C.

The following examples are included as illustrative of the process of my invention and should not be considered as limiting.

EXAMPLE 1 Germine 14,15 acetonide A suspension of p-toluene sulfonic acid monohydrate (1.90 grams) in 10 ml. of benzene is prepared and distilled at atmospheric pressure until the distillate is clear indicating that the water has been removed and that the residual benzene solution is anhydrous. The remaining benzene is removed by distillation under reduced pressure leaving as a residue 1.72 grams of anhydrous p-toluene sulfonic acid. The residue is then dissolved in 50 cc. of anhydrous ethanol and 5.09 g. of germine added. The suspension is stirred for a few minutes to dissolve the formed anhydrous germine p-toluene sulfonic acid salt. The resulting ethanol solution of germine p-toluene sulfonic acid salt is then concentrated in vacuo. The resulting residue is then redissolved in 5 ml. of ethanol and mixed with 50 ml. of anhydrous acetone to form germine acetonide p-toluene sulfonic acid salt monohydrate in substantially pure form and in high yield. The formed germine acetonide p-toluene sulfonic acid salt monohydrate is dissolved in 50 ml. ice water to which is added 50 ml. of methylene chloride. The two-phased mixture is then stirred and mixed with suflicient aqueous 25% sodium carbonate solution to adjust the pH of the reaction mixture to about 10. Stirring is continued for approximately one hour following which the methylene chloride and the aqueous layers are allowed to separate. The methylene chloride extract of product is separated, the extraction repeatedwith fresh solvent, and the extracts combined. The combined methylene chloride extract of germine acetonide free base is recovered and the methylene chloride solvent removed by evaporation leaving as a residue a substantially pure germine acetonide in quantitative yield. M.P. 250251 C., TLC-silica gel, CHCl ethanol, (9:1)-iodine vapor-single spot.

EXAMPLE 2 Germine 3,16 diacetate A solution of 8.4 grams of germine 14,15 acetonide 3,16 diacetate dissolved in 160 cc. of dilute hydrochloric acid is allowed to stand at a temperature of 25 C. for a period of 30 minutes. The solution is then neutralized by the addition of ammonium hydroxide to a pH of 8.5 while maintaining a temperature of the solution at 25 C. with cooling if necessary to produce germine 3,16 diacetate. The produced diacetate is extracted with methylene chloride and the extract than dried and evaporated under reduced pressure to produce a substantially pure crystalline product as a residue, M.P. 229-230 C. The product is recrystallized by dissolving the total amount in 70 ml. of methylene chloride and 70 cc. of petroleum ether, M.P. 242-243 C. [a] =4.5' (c.=1.238 in pyridine).

EXAMPLE 3 Germine 3-monoacetate A solution of 5.42 grams of germine 14,15 acetonide in 27 ml. of dry pyridine is prepared. To the solution is added gradually 27 cc. of acetic anhydride maintaining the temperature of the reaction mixture at 25 C. with cooling if necessary. The reaction mixture is then allowed to stand at a temperature of 25 C. for 3 /2 hours following which the entire mixture is evaporated under reduced pressure at a temperature of 40 C., leaving as a residue a mixture containing predominantly germine 14, 15 acetonide 3-monoacetate admixed with a small amount of the corresponding 3,16 diacetate. The residue obtained is flushed twice with dry benzene to remove any residual acetic anhydride. The total product obtained is dissolved in ml. methanol and heated at the reflux temperature for a period of 8 hours. The clear reaction mixture is then evaporated under reduced pressure leaving a residue comprising substantially pure, colorless crystals of germine 14,15 acetonide 3 acetate. The product obtained is flushed with dry benzene and used directly in the hydrolysis reaction.

The germine acetonide monoacetate residue is dissolved in ml. of dilute hydrochloric acid and allowed to stand at 25 C. for 30 minutes. The solution is then neutralized by the addition of ammonium hydroxide to a pH of 8.5, the germine 3-monoacetate extracted with methylene chloride and the extract dried and evaporated under reduced pressure to leave as a residue substantially pure germine 3-monoacetate which is further purified by washing with a small amount of ether. The product melts at 219220 C. [u] =+9.5 (c.=1.16 in pyridine).

What is claimed is:

1. In a process for the preparation of germine acetonide, by the reaction of germine with acetone, the improvement which comprises conducting the reaction of germine and acetone under anhydrous conditions in the presence of a hydrate-forming acid selected from the group consisting of benzene sulfonic acid, p-toluene sulfonic acid, 2,4-dinitro benzene sulfonic acid, 3,5-dinitro benzene sulfonic acid, tit-naphthalene sulfonic acid and B-naphthalene sulfonic acid to form as an intermediate product germine acetonide acid addition salt hydrate and subsequently isolating and neutralizing said acetonide acid addition salt hydrate to form germine acetonide and recovering said germine acetonide from the reaction mixture.

2. In a process for the preparation of germine 14,15 acetonide by the reaction of germine with acetone, the improvementwhich comprises conducting the reaction of germine and acetone under anhydrous conditions and in the presence of p-toluene sulfonic acid to form as an intermediate product the p-toluene sulfonic acid addition salt of germine 14,15 acetonide hydrate and subsequently isolating and neutralizing said p-toluene sulfonic acid salt of germine acetonide hydrate to form the free base germine acetonide and recovering said free base from the reaction mixture.

3. In a process for the preparation of germine acetonide by the reaction of germine with acetone, the improvement which comprises reacting together the p-toluene suifonic acid salt of germine under anhydrous conditions together with excess-acetone to form p-toluene sulfonic acid salt of germine acetonide hydrate andrecovering said acetonide hydrate from the solution.

References Cited I Jeger et al., The Alkaloids, vol. VII, 1960, Academic Press, New York,pp.405-413. i i, ALEX MAZEL, Primary Examiner J. H. TURNIPSEED, Assistant Examiner 

